Composition for accelerated production of collagen

ABSTRACT

Here the inventors provide a composition for topical application to the skin of animal comprising stearidonic acid in combination with one or more octadecatrienoic acids (CODTAs). Preferably the composition comprises  echium  oil as a source of the stearidonic acid, and a plant lipid as a source of octadcatrienoic acid, such as borage ( Borago officinalis ), wheat germ oil ( Triticum vulgare ), rosehip oil (refined;  Rosa mosqueta ),  jacaranda  oil ( Jacandra mimosa folia ), and/or  calendula  oil ( Calendula officinalis ). The composition of the invention increases collagen I secretion and thus has particular utility for promoting collagen production in skin.

FIELD OF INVENTION

This disclosure relates generally to a topical skin care composition,and more specifically to a topical skin care composition formulated toprovide accelerated production of collagen.

BACKGROUND ART

Collagen is one of the long, fibrous structural proteins whose functionsare quite different from those of globular proteins such as enzymes.Collagen is the main protein of connective tissue in animals and themost abundant protein in mammals, making up about 40% of the total. Itis tough and inextensible, with great tensile strength, and is the maincomponent of cartilage, ligaments and tendons, and the main proteincomponent of bone and teeth. Along with soft keratin, it is responsiblefor skin strength and elasticity, and its degradation leads to wrinklesthat accompany aging. Collagen strengthens blood vessels and plays arole in tissue development. Collagen is present in the cornea and lensof the eye in crystalline form. It is also used in cosmetic surgery andburn surgery.

Collagen occurs in many places throughout the body, and in manydifferent forms, each form being known as a type. There are at least 12different types of collagen, with Type I collagen being the mostabundant. The basic triple-helix structure of Type I collagen is theprototype for most of the other collagen types.

The other types of collagen differ from Type I collagen in the length oftheir triple helix and the presence or absence of globular domains attheir amino or carboxyl terminal ends. Type I collagen may be found inskin, tendons, and bone, and Types I-III are recognized as playing avital role in skin development and formation.

There is a need for skin care compositions and compositions for in-vitroadministration which enhance production of collagen.

DISCLOSURE OF THE INVENTION

The present invention in a first embodiment provides a composition fortopical application to the skin of animal especially a mammal,comprising stearidonic acid in combination with one or more plantlipids. The preferred source of stearidonic acid is a plant oil extractfrom one or more species from the Echium genus. The preferred species isEchium plantagineum L.: Purple Viper's-bugloss, Patterson's Curse.

The present invention a further embodiment provides a composition fortopical application to the skin of animal especially a mammal,comprising stearidonic acid in combination with one or more CODTA's. Thepreferred source of stearidonic acid is a plant oil extract from one ormore species from the Echium genus. The preferred species is Echiumplantagineum L.: Purple Viper's-bugloss, Patterson's Curse.

Preferably the plant lipids are those, which contain a class of fattyacids known as octadecatrienoic acids (CODTA's). Preferred CODTA's areCalendic acid, Catalpic acid, α-Eleostearic acid, Jacaric acid, andPunicic acid.

Preferred sources of the CODTA's are Calendula officinalis, Catalpaovata, Aleurites fordli, Jacandra mimosifolia and Punica granatum.

The one or more species from the Echium genus are selected from anycombination of one or more of the following:

-   -   Echium acanthocarpum Svent.    -   Echium aculeatum Poir.    -   Echium albicans Lag. & Rodr.    -   Echium angustifolium Lam.    -   Echium arenarium Guss.    -   Echium asperrimum Lam.    -   Echium auberianum Webb et Berth.    -   Echium bethencourtii Santos    -   Echium boissieri Steudel    -   Echium bonnetii Coincy    -   Echium brevirame Sprague et Hutch.    -   Echium callithyrsum Webb ex Bolle    -   Echium candicans L. fil.: Pride of Madeira    -   Echium creticutn L.    -   Echium decaisnei Webb    -   Echium flavum Desf    -   Echium gaditanum Boiss.    -   Echium gentianoides    -   Echium giganteum L.    -   Echium handiense Svent.    -   Echium humile Desf    -   Echium italicum L.: Pale Viper's-bugloss    -   Echium lancerottense Lems et Holz.    -   Echium leucophaeum Webb ex Sprague et Hutch.    -   Echium lusitanicum L.    -   Echium marianum Boiss.    -   Echium nervosum Dryand. in W.T. Aiton    -   Echium parviflorum Moench: Small-flowered Viper's-bugloss    -   Echium pavonianum Boiss.    -   Echium pininana Webb et Berth.: Giant Viper's-bugloss    -   Echium plantagineum L.: Purple Viper's-bugloss, Patterson's        Curse    -   Echium pustulatum Sibth. & Sm.    -   Echium rosulatum Lange: Lax Viper's-bugloss    -   Echium russicum J.F.Gmel.    -   Echium sabulicola Pomel    -   Echium salmanticum Lag.    -   Echium simplex DC.    -   Echium strictum L.f.    -   Echium sventenii Bramw.    -   Echium tuberculatum Hoffmanns. & Link    -   Echium virescens DC.: Tower of Jewels    -   Echium vulgare L: Viper's Bugloss    -   Echium webbii Coincy    -   Echium wildpretii Pears. ex Hook. fil.

In a further embodiment in accordance with the present invention thereis provided a method of treating skin, comprising topically applying acomposition comprising stearidonic acid in combination with one or moreplant lipids to the skin.

In a further embodiment in accordance with the present invention thereis provided a method of treating skin, comprising topically applying acomposition comprising stearidonic acid in combination with one or moreCODTA's to the skin.

In a further embodiment in accordance with the present invention thereis provided the use of stearidonic acid in combination with one or moreplant lipids, to accelerate collagen production in skin.

In a further embodiment in accordance with the present invention thereis provided the use of stearidonic acid in combination with one or moreCODTA's, to accelerate collagen production in skin.

In a further embodiment in accordance with the present invention thereis provided the use of stearidonic acid or a physiologically acceptablederivatives thereof in combination with one or more plant lipids, or aphysiologically acceptable derivative thereof for the manufacture of amedicament to accelerate collagen production in skin.

In a further embodiment in accordance with the present invention thereis provided the use of stearidonic acid or a physiologically acceptablederivatives thereof in combination with one or more CODTA's, or aphysiologically acceptable derivative thereof for the manufacture of amedicament to accelerate collagen production in skin.

A pharmaceutical composition for treating skin conditions including agedskin, comprising stearidonic acid or a physiologically acceptablederivatives thereof in combination with one or more plant lipids, or aphysiologically acceptable derivative thereof, and a physiologicallyacceptable carrier.

A pharmaceutical composition for treating skin conditions including agedskin, comprising stearidonic acid or a physiologically acceptablederivatives thereof in combination with one or more CODTA's, or aphysiologically acceptable derivative thereof, and a physiologicallyacceptable carrier.

In a preferred embodiment the echium oil is extracted from plant seedusing a supercritical carbon dioxide extraction process.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Cell viability in human dermal fibroblast cultures treated withechium oil formulated in three different diluents. Echium oil was eitherused neat, or diluted in cell culture medium, 2 mM BSA or 2 mM BSA/1.8%DMSO. Results shown are means of triplicate wells, and are expressed asa percentage of untreated control viability. The five bars for eachtreatment represent the five different volumes of each dilution tested;from left to right they are 10 (no shading), 8, 6, 3 and 1 μl (mostshaded). Cell culture medium volume in each well was 100 μl.

FIG. 2: Cell viability in human dermal fibroblast cultures treated withtest oils (first screen). Test oils were pre-diluted, 1/10, in 2 mMBSA/1.8% DMSO. Results shown are means of triplicate wells, and areexpressed as a percentage of untreated control viability. The five barsfor each treatment represent the five different volumes of each oiltested; from left to right they are 10 (no shading), 8, 6, 3 and 1 μl(most shaded). Cell culture medium volume in each well was 100 μl.

FIG. 3: Cell viability in human dermal fibroblast cultures treated withtest oils (second screen). Test oils were pre-diluted, as indicated, in2 mM BSA/1.8% DMSO. Results shown are means of triplicate wells, and areexpressed as a percentage of untreated control viability. The five barsfor each treatment represent the five different volumes of each oiltested; from left to right they are 10 (no shading), 8, 6, 3 and 1 μl(most shaded). Cell culture medium volume in each well was 100 μl

FIG. 4: Selected test oil dilutions and formulations of test oil mixes.Echium oil/test oil mixes were formulated to be added at a final volumeof 10 μl in 100 μl of cell culture medium.

FIG. 5: Effect of test oil mixes on collagen I secretion by human dermalfibroblasts. Collagen I levels were determined by ELISA (Cosmo Bio CoLtd.) and are expressed as a percentage of the levels observed insamples from cultures treated with diluent (vehicle) alone. Pepsindigests of both cell culture medium, and the cell culture surface(plate) from each treated culture well were analysed. Each barrepresents the mean of replicate samples. The coding of each test oil isshown on the right-hand side of the graph. Test oil mixes (3:1, echiumoil:test oil ratio) were formulated as detailed in FIG. 4.

FIG. 6: Test plate layouts and absorbance readings for WST-1 cellviability assay for echium oil (Epi63) dilutions. Figures representabsorbance at 450 nm minus absorbance at 620 nm. The following wells onplate 2 gave unexpectedly low readings: A6, C2, E2, E3, F2, F3, F4—thesewere in samples diluted in 2 mM BSA and were not considered to be worthrepeating, as results from samples diluted in 2 mM BSA/1.8% DMSO—thepreferred diluent—showed good consistency.

FIG. 7: Cell viability expressed as a percentage of untreated controlcell viability for cell culture wells treated with echium oil (Epi63)dilutions. Viable cell number is directly related to the correctedabsorbance readings (A_(450nm)-A_(620nm)). The mean absorbance readingfor untreated control culture wells was 1429.57.

FIG. 8: Plate layouts and absorbance readings from the firstcytotoxicity screen of test oils.

FIG. 9: Cell viability expressed as a percentage of untreated controlcell viability for cell culture wells treated with test oilsdilutions—screen I. Viable cell number is directly related to thecorrected absorbance readings (A_(450nm)-A_(620nm)). The mean absorbancereading for untreated control culture wells was 1764.17. All oils werediluted 1/10 in 2 mM BSA/1.8% DMSO.

FIG. 10: Plate layouts and absorbance readings from the secondcytotoxicity screen of test oils. Two well G1 and H1 (10 μl vehicle) onplate 1 gave very low absorbance readings indicative of no WST-1 reagentbeing added to these welts.

FIG. 11: Cell viability expressed as a percentage of untreated controlcell viability for cell culture wells treated with test oilsdilutions—screen II. Viable cell number is directly related to thecorrected absorbance readings (A_(450nm)-A_(520nm)). The mean absorbancereading for untreated control culture wells was 1235.8. All oils werediluted in 2 mM BSA/1.8% DMSO.

FIG. 12: Plate layouts and absorbance readings from the cytotoxicityscreen of echium oil/test oil mixes. Echium oil/test oil mixes wereformulated as detailed in Table 3.1; 10 μl of each oil mix were added toeach well (100 μl cell culture medium volume). Echium labelled wellswere treated with echium oil alone.

FIG. 13: Cell viability expressed as a percentage of untreated controlcell viability for cell culture wells treated with echium oil/test oilsmixes. Viable cell number is directly related to the correctedabsorbance readings (A_(450nm)-A_(620nm)). The mean absorbance readingfor untreated control culture wells was 1552.33. All oils were dilutedin 2 mM BSA/1.8% DMSO.

FIG. 14: Plate layouts and absorbance readings for ELISA determinationof collagen I. Collagen I levels were determined in pepsin digests ofcell culture medium samples (coded in yellow) and pepsin digests of thetissue culture surface (coded in green), prepared in accordance with theELISA kit manufacturer's recommendations (Cosmo Bio Co Ltd.). Collagen Istandard concentrations are also given and a plot of the Collagen Istandard curve is shown. Each sample comes from a separate cell culturewell.

FIG. 15: Collagen I concentrations in assay samples. Collagen Iconcentration are expressed in μg/ml, and were determined using theformula calculated from the standard curve of the ELISA assay. Oil mixeshighlighted in pale blue indicate samples which demonstrated >10%increase in collagen concentration relative to levels observed incultures treated with diluent alone (vehicle=2 mM BSA/1.8% DMSO), for agiven set of samples (medium or culture surface). Figures in bold arefor oil mixes that increased levels of collagen I in both culture mediaand adhered to the cell culture surface.

FIG. 16: Formulation of test oils. Rosehip (refined), borage, jacarandaand calendula oil were formulated with and without echium (base) oil, inaccordance with data obtained from the earlier study; 200 μl of eachtest oil mix was added to 2 ml culture medium. The diluent employed was2 mM BSA/1.8% DMSO—maximum DMSO achieved in culture wells was 0.18%.

FIG. 17: Levels of secreted collagen I in the culture medium of humandermal fibroblasts treated with test oils/test oil combinations. Datashown are the means of values obtained from two separate cell culturewells. Oils are referred to on the x-axis by their reference number (SeeFIG. 16). The test oil combination of refined rosehip oil (Epi29) andechium oil (Epi63) resulted in the most consistent increase in collagenI in the culture medium. Raw data may be found in FIGS. 19 to 30.

FIG. 18: Levels of secreted collagen I in the adhered to the wells ofthe culture plates, from human dermal fibroblasts treated with testoils/test oil combinations. Data shown are the means of values obtainedfrom two separate cell culture wells, Oils are referred to on the x-axisby their reference number (See FIG. 16). All test oil combinationsenhanced the levels of adhered collagen I in the cultures relative tovehicle-treated and untreated controls. With the exception of jacarandaoil (Epi60), the response of donor 2 cells to the test oils was greaterthan the response of donor 1 cells. Raw data may be found in FIGS. 19 to30.

FIG. 19: Donor 1—plate layout and raw absorbance data for media samples.Samples with the same label in adjacent columns are replicate samplesfrom the same culture well. Samples with same label in the same columnare from different culture wells. Absorbance was determined at 450 nm.

FIG. 20: Donor 1—standard curve for media samples.

FIG. 21: Donor 1—collagen I values determined from standard curve formedia samples.

FIG. 22: Donor 1—plate layout and raw absorbance data for plate (adheredcollagen) samples. Samples with the same label in adjacent columns arereplicate samples from the same culture well. Samples with same label inthe same column are from different culture wells. Absorbance wasdetermined at 450 nm.

FIG. 23: Donor 1—standard curve for plate (adhered collagen) samples.

FIG. 24: Donor 1—collagen I values determined from standard curve forplate (adhered collagen) samples.

FIG. 25: Donor 2—plate layout and raw absorbance data for media samples.Samples with the same label in adjacent columns are replicate samplesfrom the same culture well. Samples with same label in the same columnare from different culture wells. Absorbance was determined at 450 nm.

FIG. 26: Donor 2—standard curve for media samples.

FIG. 27: Donor 2—collagen I values determined from standard curve formedia samples.

FIG. 28: Donor 2—plate layout and raw absorbance data for plate (adheredcollagen) samples. Samples with the same label in adjacent columns arereplicate samples from the same culture well. Samples with same label inthe same column are from different culture wells. Absorbance wasdetermined at 450 nm.

FIG. 29: Donor 2—standard curve for plate (adhered collagen) samples.

FIG. 30: Donor 2—collagen I values determined from standard curve forplate (adhered collagen) samples.

DETAILED DESCRIPTION OF THE INVENTION

It had previously been known that stearidonic acid, and echium oil as asource of stearidonic acid, can beneficially be used to improve theappearance of skin. However the biological function of the stearidonicacid or echium oil in promoting this activity has not been determined.

Furthermore, it is also previously known that octadecatrienoic acids(CODTAs) such as Calendic acid, Catalpic acid, α-Eleostearic acid,Jacaric acid, and Punicic acid, and plant lipids as a source ofoctadecatrienoic acids, can have a role in the regeneration of skin.

Against this background, the present inventors decided to study theeffect of a combination of stearidonic acid with octadecatrienoic acidon collagen production in vitro.

Surprisingly, they determined that combinations stearidonic acid,including echium oil as a source of stearidonic acid, andoctadecatrienoic acid, including different plant lipids as a source ofoctadecatrienoic acids, increased collagen I secretion in human dermalfibroblast cultures, by increasing both the levels of soluable collagenand adhered collagen I. This ability of these compounds to promotecollagen I production had not previously been identified.

Eight test oil mixes increased the amount of measurable collagen I inpepsin digests of the cell culture surface (representing secretedcollagen that had adhered to the wall and the base of the cell culturewells containing the treated fibroblasts): rosehip (refined), borage,jacaranda, calendula, pomegranate, borage (refined), and the base,echium oil. These data are illustrated in the accompanying figures. Fouroil mixes—rosehip (refined), borage, jacaranda and calendula—can be seento have increased soluble collagen I in the cell culture medium andadhered collagen I on the cell culture surface. Moreover, Jacaric acid(an octadecatrienoic acid) in combination with the Stearidonic acid wasfound to strongly induce collagen I secretion by human dermalfibroblasts. Calendic acid (also a octadecatrienoic acid) had similarpotency to Jacaric acid. These surprising findings could not have beenpredicted, and is not obvious from, existing information regarding thesematerials. They also demonstrate that stearidonic acid andoctadecatrienoic acid can have a synergistic effect to promote collagenproduction.

By promoting collagen production, it can be expected that compositionscontaining a combination of stearidonic acid, and echium oil as a sourceof stearidonic acid, with octadecatrienoic acid, and plant lipids as asource of octadecatrienoic acid, would have much utility as topicalcosmetic formulations. Until the present invention, it had not beenappreciated that a combination of stearidonic acid, and echium oil as asource of stearidonic acid, in combination with octadecatrienoic acid,and plant lipids as a source of octadecatrienoic acid, can act topromote collagen production in the skin, and therefore have anadvantageous use as a topical formation for application to the skin.

A first aspect of the invention provides a composition for topicalapplication to the skin of animal comprising stearidonic acid incombination with one or more octadecatrienoic acids (CODTAs).

Stearidonic acid is an ω-3 essential fatty acid, sometimes calledmoroctic acid. It is biosynthesized from alpha-linolenic acid by theenzyme delta-6-desaturase. Natural sources of this fatty acid are theseed oils of hemp, blackcurrant and echium, and the cyanobacteriumspirulina. It has the chemical formula: C₁₈H₂₈O₂.

Stearidonic acid can be obtained in a chemically pure form from a numberof different suppliers. For example, Cayman Chemical(http://www.caymanchem.com/) supplies Stearidonic Acid ethyl ester asproduct number 10006856.

However, preferably for the composition of the present inventionstearidonic acid is present as part of an extract from a natural source,such as a plant or seed oil extract. Such plant or seed oil extracts arewidely available; for example, hemp oil containing stearidonic acid canbe obtained commercially from a wide variety of suppliers. Other sourcesof stearidonic acid include black current oil extract, which is againobtainable from a variety of different sources.

Preferably the composition of this aspect of the invention comprises aplant oil extract from a species from the Echium genus as the source ofthe stearidonic acid. A list of species of the Echium genus is providedable under paragraph [0010] above, from which suitable extracts can beprepared. Preferably the plant oil extract is from Echium plantagineumL.: Purple Viper's-bugloss, Patterson's Curse. Preferably the oilextract contains about 13% stearidonic acid.

Echium oil is produced from the plant seed of Echium plantagineum, alsoknown as Purple Viper's Bugloss. This oil can be obtained from a widevariety of suppliers: for example, G4/SSD Ltd (Orford Hall, BinbrbokBusiness Park, Brookenby, Market Rasen, Lincolnshire LN8 6HF). Theechium oil produced by G4/SSD Ltd has an excellent balance of essentialfatty acids, with approximately 45% n-3, including 13% stearidonic acid(SA), 25% n-6, including 10% gamma linolenic acid (GLA), and 18% n-9fatty acids. Echium oil contains two fatty acids that are not normallyfound in one singular natural seed oil, namely gamma linolenic acid(GLA) and stearidonic acid (SA). These two EFAs are vital as startingpoints in the formation of longer chain fatty acids, prostaglandins andother metabolites. Cold-pressed echium oil retains most of theantioxidants that are beneficial not only to help preserve the naturaloil, but they are also being proved to be beneficial as activeingredients in promoting good health.

In addition to a stearidonic acid, and echium oil as a source ofstearidonic acid, the composition of this aspect of the invention alsocomprises one or more octadecatrienoic acids (CODTAs).

By “octadecatrienoic acid” we include that the composition include oneor more of these types of polyunsaturated fatty acid compoundsOctadecatrienoic acids can be readily obtained from a variety ofdifferent chemical suppliers; for example, Sigma-Aldrich supplied a widerange of such compounds.

Preferably the composition comprises one or more plant lipids as thesource of the octadecatrienoic acid. By “one of more plant lipids”, weinclude that the composition includes lipids extracted from one or moredifferent plant sources. For example, a composition of the inventionwhich has plant lipid from Borago officinalis, in addition to thestearidonic acid, specifically echium oil as a source of stearidonicacid, is considered to be a composition of this aspect of the invention.

Such plant lipid can be obtained from a variety of different naturalsources. The following plant lipids are examples of those that can beused in the composition of this aspect of the invention: rosehip oil(cold pressed; organic; and refined) can be obtained from Seatons(http://www.seatons-uk.co.uk/); pomegranate oil (Seatons); Tung oil(http://www.made-in-china.com/); borage oil (including refined borageoil; Seatons); Hempseed oil (Seatons); Flax oil(http://www.bulknaturaloils.com/); wheat germ oil (Seatons); camelinaoil (Seatons); jacaranda oil (G4/SSD Ltd, details above); calendula oil(Springdale).

Preferably the plant lipid is borage oil (Borago officinalis), wheatgerm oil (Triticum vulgare) rosehip oil (refined; Rosa mosqueta),jacaranda oil (Jacandra mimosifolia), and/or calendula oil (Calendulaofficinalis).

Preferably the octadecatrienoic acid is Calendic acid, Catalpic acid,α-Eleostearic acid, Jacaric acid and/or Punicic acid.

A particularly preferred embodiment of the invention is wherein thecomposition comprises plant oil extract from Echium plantagineum L.:Purple Viper's-bugloss, Patterson's Curse, and the plant lipid is borageoil (Borago officinalis). A further particularly preferred embodiment ofthe invention is wherein the composition comprises plant oil extractfrom Echium plantagineum L.: Purple Viper's-bugloss, Patterson's Curse,and the plant lipid is wheat germ oil (Triticum vulgare).

The composition of this aspect of the invention can have range of ratiosof stearidonic acid/octadecatrienoic acid (CODTAs): for example, from10:1 to 1:10. However, preferably the ratio is about 1:1; 2:1; 3:1; 4:1or 5:1. Most preferably the ratio is about 3:1.

Also, as appropriate according to the embodiment of the composition ofthis aspect of the invention can have range of ratios of plant oilextract to plant lipid ratio: for example, from 10:1 to 1:10. However,preferably the ratio is about 1:1; 2:1; 3:1; 4:1 or 5:1 plant oilextract to plant lipid ratio. Most preferably the ratio is about 3:1.

As can be appreciated by the skilled person, the composition of thisaspect of the invention can also have a range of different quantities ofthe stearidonic acid, and echium oil as a source of stearidonic acid,and octadecatrienoic acid, and plant lipids as a source ofoctadecatrienoic acid, depending on the potency of the activeingredients in increasing the production of collagen; the amount ofcomposition to be applied; and the frequency of the application of thetopical composition to the skin.

By way of guidance, the examples section set out below providesinformation on the ratios and amounts of the echium oil extract andplant lipids that can act promote collagen production. The examples alsoprovide guidance for a series of assays to determine the effect of thecomposition on collagen production. For example, where the compositionof the invention comprises echium oil and borage oil, it is shown in theexamples that 10% echium oil and 3.33% borage oil in the finalcomposition can act to promote collagen production. For refined rosehipoil, again a 10% echium oil and 3.33% borage oil mixture can be used.For jacaranda and calendula, 1% echium oil and 0.3% plant lipid can beused.

Supercritical carbon dioxide is now well established as a solvent foruse in extraction. This is for a number of reasons. It can generallypenetrate a solid sample faster than liquid solvents because of it'shigh diffusion rates, and can rapidly transport dissolved solutes fromthe sample matrix because of it's low viscosity. There are also ofcourse less solvent residues present in the products. When used toprepare plant extracts, supercritical carbon dioxide extractionprocesses can provide an extract having less than 0.1% contamination.

The inventors have determined that plant oils and plant lipids extractedfrom source materials using supercritical carbon dioxide extractionprocesses retain greater ability to promote increased collagenproduction than oils and lipids extracted using other methods.

Hence a further preferred embodiment of this aspect of the invention iswherein the plant oil extract and plant lipid is extracted from plantseed using a supercritical carbon dioxide extraction process.Supercritical carbon dioxide extraction from natural products can beperformed by a number of different companies; for example, Sotanix(http://botanix.co.uk/index.html) and NATECO₂(http://www.nateco2.de/index.htm)

The first aspect of the invention provides a composition for topicalapplication to the skin of animal. Preferably the animal is a human.

There are a number of ways in which the composition according to thefirst aspect of the invention can be formulated for topicaladministration to the skin of an animal.

Thus, for example, the composition may be in the form of a powder,liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle,transdermal patch, liposome or any other suitable form that allows forthe composition to be topically administered. The formulation maytherefore be one that can be applied by spreading or by spraying ontothe skin. It will be appreciated that the vehicle of the composition ofthe invention should be one which is well tolerated by the subject towhom it is given. The formulation can also contain further ingredients,such as perfumes, colorants, preservatives, or further biologicallyactive ingredients.

For application topically to the skin, the composition of the inventioncan be formulated as a suitable ointment containing the activeingredients suspended or dissolved in, for example, a mixture with oneor more of the following: mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound,emulsifying wax and water. Alternatively, they can be formulated as asuitable lotion or cream, suspended or dissolved in, for example, amixture of one or more of the following: mineral oil, sorbitanmonostearate, a polyethylene glycol, liquid paraffin, polysorbate 60,cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol andwater.

However it is preferred that the composition of the invention isformulated as particles in an emulsion gel. It is thought that such aformulation will beneficially allow the composition of the invention tobe solubilised to facilitate effective delivery, especially to the skin.Preferred formulation technology include the ‘nanoemulsions’ prepared byMalvern Cosmeceutics Limited (http://www.malceutics.com/index.htmi).

A further aspect of the invention provides a pharmaceutical compositioncomprising a composition according to the first aspect of the inventionand a pharmaceutically acceptable excipient. A “pharmaceuticallyacceptable vehicle” as referred to herein is any physiological vehicleknown to those of ordinary skill in the art useful in formulatingpharmaceutical compositions.

A further aspect of the invention provides a method of treating skincomprising topically applying a composition or a pharmaceuticalcomposition according to the previous aspects of the invention to theskin.

A further aspect of the invention provides a composition or apharmaceutical composition according to the previous aspects of theinvention for increasing collagen production in skin.

A further aspect of the invention provides a composition or apharmaceutical composition according to the previous aspects of theinvention use as a medicament for increasing collagen production inskin.

A preferred embodiment of each of the above further aspects of theinvention is where the composition comprises plant oil extract fromEchium plantagineum L.: Purple Viper's-bugloss, Patterson's Curse, andthe plant lipid is borage oil (Borago officinalis) and/or wheat germ oil(Triticum vulgare).

EXPERIMENTAL EXAMPLES Example 1 An In Vitro Study to Determine theEffect of Applied Lipids on Fibroblast Collagen Synthesis

Experiments were undertaken to determine the efficacy of plant-derivedlipids use in combination with echium oil in promoting collagen Isynthesis in human dermal fibroblast cultures. Human dermal fibroblastcultures derived from explant cultures of human skin, followingenzymatic removal of the epidermis, provide a useful model for skintoxicity screening, determining the efficacy of UV protective agents andstudying agents that may modify gene/protein expression within thedermis. This study was performed in order to determine the efficacy of arange of plant-derived oils in promoting collagen I secretion by dermalfibroblasts. Secreted collagen I was assayed in pepsin digests of thecell culture medium and pepsin digests of the cell culture surface byELISA.

Experimental Methods:

Human dermal fibroblasts (originally derived from explant cultures ofnormal human breast dermis) were cultured under standard conditions. Arange of test oils supplied by the client were initially screened forcytotoxicity using a 96-well format, formazan-based, cell viabilityassay; this assay employed a single dose of test oil, with viabilitybeing measured after 48 hours. From this assay, a maximum tolerateddilution of each test oil was chosen. Subsequently, mixtures of the testoils with echium oil (which was chosen to be the base oil) were preparedat a ratio of one part test oil to three parts echium oil. These testoil/echium oil mixes were also screened for cytotoxicity, prior to beingemployed in a 6 well plate format assay. Cells were exposed to the oilmixes for a period of three days. Cell culture media, cells and theculture plates were all harvested and stored at −80° C., prior toanalysis. A commercial ELISA kit was employed in order to determine theconcentration of collagen I in the culture medium and the amount ofsecreted collagen that had adhered to the cell culture well.

Detailed Methods:

Culture of Human Dermal Fibroblasts

Human dermal fibroblasts were maintained in DMEM, with 10% FCS,L-glutamine (2 mM), penicillin (100 IU/ml) and streptomycin (100 μg/ml),at 37° C., in 95% air/5% CO₂ atmosphere, with 95% relative humidity. Foruse in cytotoxicity and collagen I assays, cells were harvested bytrypsin/EDTA treatment.

Test Agents

The test agents were supplied in liquid form, at room temperature, bythe client. The test agents are listed in Table 1 below together withtheir reference code.

TABLE 1 Test agent Code Echium oil Epi63 Rosehip oil - cold pressedEpi27 Rosehip oil - organic Epi28 Rosehip oil - refined Epi29Pomegranate Epi30 Tung Epi31 Borage Epi35 Hemp Epi36 Flax Epi37Wheatgerm Epi38 Carmelina Epi39 Borage - refined Epi40 Echium seed Epi41Jacaranda Epi60 Calendula Epi61

Cytotoxicity Screening of Test Agents.

Human dermal fibroblasts were plated at 2×10⁴ cells per well (100 μlvolume) in 96 well plates. After 48 hours, the medium was changed andwells were treated with test oils, either alone or in combination withechium oil. Cells were exposed to the oils for 48 hours; at the end ofthis period, viable cell number was determined using WST-1 Quick-cellproliferation assay (BioVision Inc.). For the WST-1 assay, the mediumwas refreshed in each culture well prior to the addition of 10 μl ofassay reagent. Cells were incubated for 2 hours at 37° C., after whichtime the absorbance at 450 nm and 620 nm was determined using an AnthosH III plate reader.

Determination of Collagen I Secretion

Cells were seeded at 5×10⁵ cells/well in 6 well plates. After 24 hours,the medium was changed and cells were treated with test oil mixes. Cellswere exposed to test oil mixes for 72 hours, with medium and test oilsbeing refreshed daily. At the end of the exposure period, the medium washarvested from the cell culture wells and stored at −80° C. The cellswere harvested from the plate by scraping with a rubber policeman, andboth the harvested cells and the culture dish were stored at −80° C.,also. Collagen I secretion was determined by ELISA (Cosmo Bio Co. Ltd.,Japan), using pepsin digests of both the cell culture media samples andthe cell culture well surface. ELISA and pepsin digestions were carriedout according to the manufacturer's instructions, with absorbance at 450nm being determined using an Anthos H Ill plate reader.

Results:

The base, echium oil was found to be well tolerated by the dermalfibroblasts at a final dilution of 1/100—a vehicle of 2 mM BSA/1.8% DMSOwas chosen as a diluent. Subsequently, all test oils were assayed forcytotoxicity at an initial dilution of 1/100-1/1000. Ten of the testoils were tolerated within dilution; these were rosehip (cold pressed),rosehip (refined), pomegranate, borage, hemp, flax, wheatgerm,carmelina, borage (refined) and echium seed. Four oils required furtherdilution in the range 1/1000-10,000 in order to find a toleratedconcentration; these oils were rosehip (organic), tung, jacaranda andcalendula. Dilutions of echium oil and each test oil were thencalculated to provide echium/test oil mixes with a 3:1 ratio, which didnot exceed the tolerated concentration of either test or echium oil whenadded to the cell cultures. It was demonstrated that all the echium/testoil mixes had no associated cytotoxicity, and so they were all tested inthe three day exposure assay to determine their effects on collagen Isecretion. Four echium/test oil mixes increased collagen I in both thecell culture medium and the amount of collagen adhered to the cellculture plates; the test oils were rosehip (refined), borage, jacarandaand calendula). Three other test oils were associated with increases incollagen I in the culture medium (rosehip—cold pressed, tung andwheatgerm); four oils increased the amount of collagen adhered to thecell culture well (pomegranate, echium, borage—refined and echium seed).Three test oils had little effect on collagen I secretion(rosehip—organic, hemp and flax), and one resulted in an apparentreduction in collagen I secretion (carmelina).

Results and Discussion Echium oil was well tolerated by human dermalfibroblasts at dilutions up to 1/100 Echium oil was either added neat tocell culture wells, or formulated in one of three diluents—normal cellculture medium, 2 mM BSA and 2 mM BSA/1.8% DMSO; BSA was chosen as itcan act as a carrier molecule and DMSO enhances cellular uptake.Dilutions of 1/10, 1/100 and 1/1000 were prepared in each diluent. Arange of volumes of each echium oil dilution were tested: 10, 8, 6, 3,and 1 μl were added to triplicate wells containing 100 μl of cellculture medium.

FIG. 1 shows the relative viable cell number in fibroblast culturestreated with the different echium oil preparations. Echium dilutions of1/10 in all diluents were well tolerated, with viabilities within theexpected range. Neat echium oil was toxic to the fibroblasts.

From these results it was decided that the 2 mM BSA/1.8% DMSO diluentwas suitable for use, in combination with the echium oil. This diluentwas selected in preference as it had the potential to enhance uptake ofthe oil by the fibroblast cells, and was used as a diluent in thescreening of the other, individual oils. It was also decided that 10 μlof the 1/10 dilution of the echium oil (equivalent to 1/100 finaldilution) would be set as the maximum permitted dose of echium oil inthe subsequent assay of test oil mixes.

Test oils demonstrated a wide range of tolerability. Dilutions of 1/10in 2 mM BSA/1.8% DMSO were prepared for all test oils and used for aninitial cytotoxicity screen, with diluted test oil volumes of 1-10 μlbeing added to cell culture wells containing 100 μl of medium (givingfinal dilution in the range of 1/100 to 1/1000). If test oilsdemonstrated cytotoxicity over this dilution range, the screen wasrepeat using test oil pre-diluted at 1/100.

The results from the test oil screen are shown in FIG. 2. The majorityof the test oils were tolerated within the initial dilution range thatwas tested; however six oils were re-tested in order to confirm atolerated dilution: these oils were rosehip (organic), pomegranate,tung, wheatgerm, jacaranda and calendula. The data from this secondround of screening are shown in FIG. 3. The two rounds of screening weresufficient to select a tolerated volume of each test oil. These datawere then used to calculate echium/test oil combination volumes thatwere to be assayed for their effect on collagen I secretion. Wherepossible, a conservative approach was used in deciding the appropriatedilution of test oil to select.

The tolerated dilution of test oils and the volumes used in making testoil mixes, using a set ratio of 3 parts echium oil to load test oil areshown in FIG. 4. It can be seen from FIG. 4 that for some test oil mixesthe final dilution of echium oil that could be achieved in a cellculture cell (100 μl volume) was less than 1/100 (the maximum tolerateddilution of echium oil), due to the limiting cytotoxicity of the testoil; these test oil mixes were with rosehip (organic), rosehip(cold-pressed), tung, jacaranda and calendula.

All test oil mixes were subject to a final round of cytotoxicityscreening. All were found to be non toxic (data may be found in FIGS. 6to 15). FIG. 3: Cell viability in human dermal fibroblast culturestreated with test oils (first screen).

Test oils were pre-diluted, 1/10, in 2 mM BSA/1.8% DMSO. Results shownare means of triplicate wells, and are expressed as a percentage ofuntreated control viability. The five bars for each treatment representthe five different volumes of each oil tested; from left to right theyare 10 (no shading), 8, 6, 3 and 1 μl (most shaded). Cell culture mediumvolume in each well was 100 μl. FIG. 3: Cell viability in human dermalfibroblast cultures treated with test oils (second screen).

Test oils were pre-diluted, as indicated, in 2 mM BSA/1.8% DMSO. Resultsshown are means of triplicate wells, and are expressed as a percentageof untreated control viability. The five bars for each treatmentrepresent the five different volumes of each oil tested; from left toright they are 10 (no shading), 8, 6, 3 and 1 μl (most shaded). Cellculture medium volume in each well was 100 μl. FIG. 4: Selected test oildilutions and formulations of test oil mixes. Echium oil/test oil mixeswere formulated to be added at a final volume of 10 μl in 100 μl of cellculture medium.

Rosehip (refined), borage, jacaranda and calendula test oil mixes allincreased collagen I secretion by human dermal fibroblasts. ELISAanalysis of pepsin digests of cell culture media samples demonstratedthat 7 test oil mixes increased the amount of measurable secretedcollagen I, relative to levels from cultures treated: these oils wererosehip (refined), borage, jacaranda, calendula, rosehip (cold-pressed),tung and wheatgerm. Eight test oil mixes increased the amount ofmeasurable collagen I in pepsin digests of the cell culture surface(representing secreted collagen that had adhered to the wall and thebase of the cell culture wells containing the treated fibroblasts):rosehip (refined), borage, jacaranda, calendula, pomegranate, borage(refined), echium seed and the base, echium oil.

These data are illustrated in FIG. 5. Four oil mixes—rosehip (refined),borage, jacaranda and calendula—can be seen to have increased solublecollagen I in the cell culture medium and adhered collagen I on the cellculture surface. Of these four oil mixes, jacaranda showed the greatestefficacy; the jacaranda and calendula mixes were also very potent, withfinal test oil dilutions of 3/10,000 and echium dilutions of 9/10,000being achieved in the cell culture wells.

The data obtained also showed that treatment with the diluent itself (2mM BSA/1.8% DMSO) increase collagen I secretion, relative to levelsobserved in untreated control cultures. This may simply reflect thediluents ability to increase the uptake of media components thatpositively regulate collagen I secretion.

FIG. 5: Effect of test oil mixes on collagen I secretion by human dermalfibroblasts. Collagen I levels were determined by ELISA (Cosmo Bio CoLtd.) and are expressed as a percentage of the levels observed insamples from cultures treated with diluent (vehicle) alone. Pepsindigests of both cell culture medium, and the cell culture surface(plate) from each treated culture well were analysed. Each barrepresents the mean of replicate samples. The coding of each test oil isshown on the right-hand side of the graph. Test oil mixes (3:1, echiumoil:test oil ratio) were formulated as detailed in FIG. 4.

Conclusions Jacaranda oil, in combination with the base, echium oil, wasfound to strongly induce collagen I secretion by human dermalfibroblasts, under the culture conditions employed in this study.Calendula oil had similar potency to jacaranda oil, although itsefficacy at increasing collagen I secretion was not so great. Rosehip(refined) and borage had similar efficacy to calendula, although theywere tested at a ten-fold higher final concentration. Treatment withthese four oils was associated with increased collagen I in both thecell culture medium, and that adhered to the cell culture well. Otheroils were associated with increases in one or the other of these twoparameters.

Example 2 An Investigation of the Ability of Applied Lipids to IncreaseCollagen Secretion by Human Fibroblasts In Vitro

Summary

To confirm the ability of test oil and test oil combinations to enhancelevels of collagen in cultures of human dermal fibroblasts.

Previously, a range of test oils were screened for cytotoxicity andtheir ability to increase levels of collagen in cultures of human dermalfibroblasts. In this study the test oils that had demonstrated greatestefficacy in increasing levels of measurable collagen were re-tested,including borage, refined rosehip, jacaranda and calendula oils; theseoils were tested alone and in combination with the base oil, echium.Both adhered collagen and collagen in the tissue culture medium wereassayed. Two sets of human dermal fibroblast cultures, derived fromdonor skin from two separate individuals, were used in this study.

The data obtained confirmed that the test oil/echium oil combinationsincreased the amount of measurable collagen in the human fibroblastcultures. This effect was observed in samples treated with vehiclealone, but as the vehicle was also able to increase collagen levels inthe culture medium this is not so surprising.

The ability of the test oils to increase the levels of extracellularcollagen in human dermal fibroblast cultures in vitro was confirmed.There was some variation in the response profile of fibroblasts derivedfrom different skin donors, but all the oils demonstrated an ability toincrease the levels of adhered collagen, relative to vehicle-treated anduntreated control cultures.

Introduction

Human dermal fibroblast cultures derived from explant cultures of humanskin, following enzymatic removal of the epidermis, provide a usefulmodel for skin toxicity screening, determining the efficacy of UVprotective agents and studying agents that may modify gene/proteinexpression within the dermis. In a previous study, dermal fibroblastcultures were employed to examine the effect of test items on collagensecretion. Secreted collagen may be found in the culture media and alsoadheres to the culture vessel/well in which the cells are grown. Acollagen I-specific ELISA can be employed to quantify collagen levels inboth these compartments.

Procedures

Culture of Human Dermal Fibroblasts

Human dermal fibroblast cultures were established from breast skinsamples obtained from two separate donors undergoing surgical proceduresfor cosmetic purposes. Dermal fibroblasts were maintained in DMEM, with10% FCS, L-glutamine (2 mM), penicillin (100 IU/ml) and streptomycin(100 μg/ml), at 37° C., in 95% air/5% CO₂ atmosphere, with 95% relativehumidity. For collagen I assays, cells were harvested by trypsin/EDTAtreatment.

Treatment of Fibroblast Cultures with Test Items

The test items were supplied in liquid form, at room temperature, by theclient. Test items were formulated according to the details in FIG. 16.Cells were seeded at 5×10⁵ cells/well in 6 well plates. After 24 hours,the medium was changed and cells were treated with test oil mixes, whichwere added at a final volume of 200 μl in 2 ml of culture medium. Alltreatments were tested on fibroblast cultures derived from skin samplesfrom two donors. Cells were exposed to test oil mixes for 72 hours, withmedium and test oils being refreshed daily. Four culture wells weretreated with each test item or test item combination, or were treatedwith vehicle, or left untreated. At the end of the exposure period, themedium was harvested from all the cell culture wells, transferred to asterile 7 ml tube and stored at −80° C. The cells were harvested fromtwo culture wells for each treatment by scraping with a rubberpoliceman, and transferred to a sterile 1.5 ml tube. The harvested cellsand the culture dish were stored at −80° C.

Determination of Secreted Collagen Levels in Fibroblast Cultures

Collagen I secretion was determined by ELISA (Cosmo Bio Co. Ltd.,Japan), using pepsin digests of both the cell culture media samples andthe cell culture well surface. ELISA and pepsin digestions were carriedout according to the manufacturer's instructions, with absorbance at 450nm being determined using an Anthos H III plate reader.

Results and Discussion

Dermal Fibroblast Cultures, Established from Skin Derived from TwoDifferent Donors, Demonstrated Increased Levels of Secreted Collagen IFollowing Treatment with Test Oils

Dermal fibroblast cultures derived from both donor 1 and donor 2demonstrated increased levels of secreted collagen I following treatmentwith test oils. There was some donor variation in the magnitude of theresponse to each treatment, and in the treatments that demonstrated thegreatest efficacy (particularly for the levels of measurable collagen inthe culture medium). Such variation in the biological responses of cellsderived from different skin donors is to be expected. The mostconsistent feature was that all oils/oil combinations increased thelevels of adhered collagen I in the culture wells, relative to bothvehicle-treated and untreated controls. Data for the levels of collagenin the media compartment and the adhered collagen compartment are shownin FIGS. 17 and 18.

Conclusions

The effect of test oils in increasing the levels of secreted collagen Iin cultures of dermal fibroblasts has been confirmed.

Example 3 Preliminary Report on a Study to Further Establish The Rolesof CODTA's when Combined with SDA in Collagen Production in Human Skin

This example provides outline results of the effect of two test activeingredient mixes on collagen I secretion by human dermal fibroblasts.Human dermal fibroblasts (originally derived from explant cultures ofnormal human breast dermis) were cultured under standard conditions. Twotest combinations of actives (CODTA's when combined with SDA) wereinitially screened for cytotoxicity before testing.

Collagen I levels were determined and are expressed as a percentage ofthe levels observed in samples from cultures treated with diluent(vehicle) alone.

Pepsin digests of both cell culture medium, and the cell culture surface(plate) from each treated culture well were analysed.

Jacaric Acid in combination with the Stearidonic Acid base was found tostrongly induce collagen I secretion by human dermal fibroblasts underthe culture conditions employed. Percentage increase 278%

Calendic Acid had similar potency to Jacaric Acid although its efficacyat increasing collagen I secretion was not so evident. Percentageincrease 187%

The invention is not restricted to the details of the foregoingembodiment(s). The invention extends to any novel one, or any novelcombination, of the features disclosed in this specification (includingany accompanying claims, abstract and drawings), or to any novel one, orany novel combination, of the steps of any method or process sodisclosed.

1. A composition for topical application to the skin of animalcomprising echium oil containing stearidonic acid in combination withone or more of jacaranda oil and calendula oil containing one or moreoctadecatrienoic acids, wherein the composition is effective to increasecollagen I production in skin.
 2. The composition of claim 1 wherein theechium oil is extracted from Echium plantagineum L.
 3. The compositionof claim 1 wherein the one or more octadecatrienoic acids is Calendicacid and/or Jacaric acid.
 4. The composition of claim 1 wherein theratio of echium oil to jacaranda oil and/or calendula oil is in therange of 10:1 to 1:10.
 5. The composition of claim 1 wherein the echiumoil and the one or more of jacaranda oil and calendula oil are extractedfrom plant seed using a supercritical carbon dioxide extraction process.6. A pharmaceutical composition comprising a composition according toclaim 1 and a pharmaceutically acceptable excipient.
 7. A method oftreating skin comprising topically applying a composition or apharmaceutical composition according to claim 1 to the skin.
 8. Acomposition or a pharmaceutical composition according to claim 1 to theskin for use as a medicament for increasing collagen production in skin.9. A composition for topical application to the skin of an animalcomprising echium oil containing stearidonic acid in combination withone or more plant lipids containing octadecatrienoic acids selected fromcalendic acid and jacaric acid, wherein the one or more plant lipids areselected from the group consisting of jacaranda oil and calendula oil,wherein the composition is effective to increase collagen I productionin skin.
 10. The composition of claim 1 wherein the ratio of echium oilto jacaranda oil and/or calendula oil is 1:5.
 11. The composition ofclaim 1 wherein the ratio of echium oil to jacaranda oil and/orcalendula oil is 1:3.
 12. The composition of claim 1 wherein the ratioof echium oil to jacaranda oil and/or calendula oil is 1:1.
 13. Thecomposition of claim 1 wherein the ratio of echium oil to jacaranda oiland/or calendula oil is 3:1.
 14. The composition of claim 1 wherein theratio of echium oil to jacaranda oil and/or calendula oil is 5:1.